Zhou, C.; Brown, M.; Blaxter, M.; The Darwin Tree of Life Project Consortium, ; McCarthy, S. A.; Durbin, R.

Plant organelle genomes, particularly the large mitochondrial genomes with intricate repetitive structures, present significant challenges for assembly. The advent of long-read sequencing technologies provides a transformative opportunity to generate complete genomes, but problems of resolving alternative structures remain. Here we introduce a novel tool for plant organelle genome assembly from high-accuracy long reads. Our method employs a k-mer based assembler for rapid assembly graph construction, integrates a profile HMM gene database for robust organelle sequence annotation, and leverages a new search method to find the best supported path through the assembly graph. We describe high-quality organelle assemblies for 195 plant species and demonstrate improvements over other methods. The assembled genomes provide multiple insights into structural complexity, heteroplasmy, and DNA exchange between organelles.